Several documents are cited throughout the text of this specification. The disclosure content of the documents cited therein (including manufacture's specifications, instructions, etc.) is herewith incorporated by reference.
Any combination of steps (including single steps only) carried out in vitro and cited through this specification can also be carried out with cell extracts or in vivo.
The present invention is exemplified using DNA methyltransferases (MTases). However, it can also be used with RNA and protein methyltransferases as well as methyltransferases acting on other biomolecules.
DNA methylation is found in almost all organisms (Jeltsch, (2002) ChemBioChem 3, 275-293). The DNA can contain the methylated nucleobases 5-methylcytosine (5-mCyt), N4-methylcytosine (4-mCyt) or N6-methyladenine (6-mAde) in addition to cytosine, adenine, thymine and guanine. These methylated nucleobases are formed by DNA methyltransferases (MTases) which catalyze the transfer of the activated methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the C5 carbon of cytosine, the N4 nitrogen of cytosine or the N6 nitrogen of adenine within their DNA recognition sequences (Cheng, (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 293-318). Since a particular nucleotide sequence may exist in its methylated or unmethylated form, DNA methylation can be regarded as an increase of the information content of DNA, which serves a wide variety of biological functions. In prokaryotes DNA methylation is involved in protection of the host genome from endogenous restriction endonucleases, DNA mismatch repair, regulation of gene expression and DNA replication. In eukaryotes DNA methylation plays a role in important regulatory processes such as gene silencing (Bird, (2002) Genes Dev. 16, 6-21), genomic imprinting (Feil and Khosla, (1999) Trends Genet. 15, 431-435), X-chromosome inactivation (Panning and Jaenisch, (1998) Cell 93, 305-308), silencing of intragenomic parasites (Yoder, (1997) Trends Genet. 13, 335-340), and carcinogenesis (Baylin, (1998) Adv. Cancer Res. 72, 141-196; Jones and Laird, (1999) Nat. Genet. 21, 163-167).
Recently, a newly designed fluorescent cofactor for the DNA adenine-6 methyltransferase from Thermus aquaticus (M.TaqI) has been presented (Pljevaljcic et al., (2003) J. Am. Chem. Soc. 125, 3486-3492). Naturally, M.TaqI catalyzes the nucleophilic attack of the exocyclic amino group of adenine within the double-stranded, palindromic 5′-TCGA-3′ DNA sequence onto the methyl group of the cofactor S-adenosyl-L-methionine (SAM or AdoMet) leading to sequence- and base-specific methyl group transfer. M.TaqI, like other DNA methyltransferases can only transfer one methyl group to each target base and DNA with a fully methylated recognition sequence is not further modified. Replacement of the methionine side chain of the natural cofactor S-adenosyl-L-methionine (SAM or AdoMet) by an aziridinyl residue leads to M.TaqI-catalyzed nucleophilic ring opening and coupling of the whole nucleoside to the target adenine in DNA. The adenosyl moiety is the molecular anchor for cofactor binding. Attachment of a fluorophore via a flexible linker to the 8 position of the adenosyl moiety does not block cofactor binding. This cofactor, 8-amino[1″-(N′-dansyl)-4″-aminobutyl]-5′-(1-aziridinyl)-5′-deoxyadenosine, can be used to sequence-specifically label DNA in a M.TaqI-catalyzed reaction (Pljevaljcic et al., (2003) J. Am. Chem. Soc. 125, 3486-3492).
The prior art describes the above mentioned N-adenosylaziridine derivative as well as 8-amino[1″-(N″-biotinyl)-4″-aminobutyl]-5′-(1-aziridinyl)-5′-deoxyadenosine (Pljevaljcic et al., (2004) Methods Mol. Biol. 283, 145-161) which can be used for labeling biomolecules (Pljevaljcic et al., (2004) ChemBioChem 5, 265-269). Labeling is carried out by using S-adenosyl-L-methionine-dependent methyltransferases. These enzymes naturally catalyze the transfer of the activated methyl group from the cofactor S-adenosyl-L-methionine (1, SAM or AdoMet) to defined nucleophilic positions within various substrates like DNA, RNA, proteins and other biomolecules leading to methylated biomolecules and the demethylated cofactor S-adenosyl-L-homocysteine (2) (Scheme 1). The ability of methyltransferases to catalyze sequence-specific, covalent modifications of biopolymers makes them interesting tools for biotechnology in general and it would be desirable to transfer, in addition to the methyl group, larger chemical entities with additional functionalities to the target biomolecules. In principle, this can be achieved with the N-adenosylaziridine derivatives mentioned above. However, the aziridine cofactors have the disadvantage that they serve as suicide substrates for methyltransferases. After coupling of the aziridine cofactor with the target biomolecule the methyltransferases can not dissociate easily and remain bound to their coupling products. Thus, the methyltransferases do not act as true catalysts and have to be employed in stoichiometric amounts with respect to the target biomolecules. In principle this limitation can be overcome by replacing the methyl group of S-adenosyl-L-methionine (1, SAM or AdoMet) with larger aliphatic groups. Studies by Schlenk (Schlenk and Dainko, (1975) Biochim. Biophy. Acta 385, 312-323; Schlenk, (1977) in Biochem. Adenosylmethionine (eds. Salvatore, Borek and Zappia), Columbia University Press, 3-17) indicated that larger chemical groups like ethyl and propyl can be transferred from S-adenosyl-L-ethionine (3) and S-adenosyl-L-propionine (4) by methyltransferases (Scheme 1). However, it was also found that enzymatic alkyl transfer rates decline drastically with increasing size of the transferable group (methyl >>ethyl>n-propyl group). This general trend was also obtained with different DNA methyltransferases (Example 3).

The drastic decline of transfer rates when using transferable groups with increased lengths (see above) prevents S-adenosyl-L-methionine derivatives from being used as effective cofactors of S-adenosyl-L-methionine-dependent methyltransferases. Since S-adenosyl-L-methionine derivatives are closely related to the natural substrates of S-adenosyl-L-methionine-dependent methyltransferases, it would be desirable to develop S-adenosyl-L-methionine-based derivatives with improved transfer rates of sulfonium bound side chains.